Analysis of GLUT4 localisation and trafficking in heart muscle cells has been achieved by a novel method using a transgenic mouse line which expresses GLUT4, with both a C-terminal GFP tag and an exofacial HA tag (HA-GLUT4-GFP), under the control of a muscle specific promoter. Cardiomyocytes were isolated by Langendorf perfusion and collagenase digestion of whole heart tissue. Cardiomyocytes were treated with insulin or oxidative metabolism stress, or electrically induced to contract, and the localisation and trafficking of HA-GLUT4-GFP analysed by immunofluorescence. The exofacial HA tag enabled fluorescent labelling of only GLUT4 exposed at the external surface. The results show that HA-GLUT4-GFP is totally excluded from the sarcolemma under basal conditions, and is trafficked to the sarcolemma when cardiomyocytes are treated with 30 nM insulin, stimulated to contract, and upon induction of metabolic stress by incubation in hypoxic buffer. The exofacial tag was only apparent at the sarcolemma and not at the t-tubule membrane using detection by whole anti-HA antibody. To monitor the internalization process, surface GLUT4 was pulse-labelled with anti-HA antibody after stimulating the cells. The stimulus was subsequently removed, and cells incubated to allow internalization of GLUT4. Anti-HA antibody-labelled GLUT4 colocalaises with clathrin at puncta at the sarcolemma, indicating that in cells returning to a basal state, GLUT4 is removed from the sarcolemma by a clathrin-mediated route. We also observed colocalisation of GLUT4 with clathrin under basal conditions at the sarcolemma. Full internalization of anti-HA antibody labelled-GLUT4 from the sarcolemma was achieved and GLUT4 detected in a predominantly a peri-nuclear compartment; indistinguishable between the specific initial stimuli. These results taken toegther imply a common pathway for internalization independent of the initial stimulus.